Metabolic profiling of Vitex Pubescens Vahl bark via UPLC-ESI-QTOF/MS/MS analysis and evaluation of its antioxidant and acetylcholinesterase inhibitory activities

Background Alzheimer’s disease is a neurodegenerative age-related disease that primarily affects the elderly population leading to progressive memory impairments and neural deficits. It is counted as a major cause of geriatric dependency and disability. The pathogenesis of Alzheimer’s disease incidence is complex and involves various hypotheses, including the cholinergic hypothesis, deposition of β-amyloid plaques, neuroinflammation, oxidative stress, and apoptosis. Conventional treatments such as donepezil aim to delay the symptoms but do not affect the progression of the disease and may cause serious side effects like hepatoxicity. The use of natural candidates for Alzheimer’s disease treatment has drawn the attention of many researchers as it offers a multitargeted approach. Methods This current study investigates the metabolic profiles of total defatted methanolic extract of Vitex pubescens bark and its polar fractions, viz. ethyl acetate and n-butanol, using ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry(UPLC-ESI-QTOF/MS/MS) technique as well as evaluate the antioxidant using free radical scavenging assays, viz. DPPH and ABTS assays and in-vitro acetylcholinesterase inhibitory activities using Ellman’s microplate assay. Results Metabolic profiling revealed a total of 71, 43, and 55 metabolites tentatively identified in the defatted methanolic extract, ethyl acetate, and n-butanol fractions, respectively. Phenolic acids were the most abundant class, viz. benzoic acids, and acyl quinic acid derivatives followed by flavonoids exemplified mainly by luteolin-C-glycosides and apigenin-C-glycosides. Quantification of the total phenolic and flavonoid contents in the total defatted methanolic extract confirmed its enrichment with phenolics and flavonoids equivalent to 138.61 ± 9.39 µg gallic acid/mg extract and 119.63 ± 4.62 µg rutin/mg extract, respectively. Moreover, the total defatted methanolic extract exhibited promising antioxidant activity confirmed through DPPH and ABTS assays with a 50% inhibitory concentration (IC50) value equivalent to 52.79 ± 2.16 µg/mL and 10.02 ± µg/mL, respectively. The inhibitory activity of acetylcholine esterase (AchE) was assessed using in-vitro Ellman’s colorimetric assay, the total defatted methanolic extract, ethyl acetate, and n-butanol fractions exhibited IC50 values of 52.9, 15.1 and 108.8 µg/mL that they proved the significant inhibition of AchE activity. Conclusion The results obtained herein unraveled the potential use of the total methanolic extract of Vitex pubescens bark and its polar fractions as natural candidates for controlling Alzheimer’s disease progression. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-024-04520-3.


Introduction
Alzheimer's disease (AD) is an irreversible neurodegenerative disorder marked by gradual and progressive memory loss, compromised cognitive and neuronal dysfunction, with subsequent deteriorated behaviorrelated skills [1].It mainly affects the elderly population over 65 years [2].The World Health Organization [3] recorded Alzheimer's disease as the seventh leading cause of mortality worldwide.It is estimated that more than 55 million people are currently diagnosed with AD, and the prevalence is speculated to surge to 78 million by 2030 [3].AD is regarded as one of the major contributors to geriatric dependency, which globally increases healthcare's economic burden [4].The pathophysiology of AD is sophisticated with multifactorial hypotheses encompassing the cholinergic hypothesis and deposition of extracellular amyloid β (Aβ) plaques alongside other causative factors such as oxidative stress, neuroinflammation, and apoptosis [2,5].Firstly, the cholinergic hypothesis is explained by the progressive decline in the acetylcholine (Ach) neurotransmitter which is responsible for neuronal activity, plasticity, and network connectivity.This major concern in acetylcholine level occurs due to the degeneration of cholinergic neurons, appearance of cholinergic lesions, or overactivity of acetylcholinesterase enzyme (AchE) that subsequently leads to the depletion of the level of Ach [6,7].Secondly, the deposition of Aβ plaques is one of the hallmarks of Alzheimer's disease pathogenesis that causes brain structure abnormalities [2,8].The main triggering factor of neurodegenerative diseases is increasing oxidative stress [9].The brain has a plentiful amount of polyunsaturated fatty acids besides the presence of aerobic media, transition metals, and the reduction in antioxidant enzymatic activity that leads to an imbalance in the redox system, accumulation of oxygen reactive species (ROS), increased lipid peroxidation and DNA oxidation and eventually oxidative damage [1,10].Many predisposing factors as the accumulation of ROS and nitrogen-reactive species (NRS) impair the activity of mitochondria and activate the apoptotic mediators that contribute to the progressive degeneration of neurons [1].Conventional therapies improve the symptoms but have not shown any effect on delaying the disease progression [2].The current conventional therapeutic class commonly prescribed is acetylcholinesterase inhibitors as donepezil, galantamine, and rivastigmine which depend on restoring Ach level by inhibiting the AchE degradable enzyme [5].Unfortunately, detrimental adverse effects such as gastrointestinal illnesses and hepatotoxicity are considered a driving cause for discovering new entities for the prevention or treatment of AD [11].Owing to the complexity of AD, some modern therapeutic strategies offer a multitargeted approach for treating neurodegenerative disorders that could be achieved through natural herbal products that act on various mechanisms of pathogenesis [5,[12][13][14].The potent antioxidant activity of some phytochemical classes, viz.polyphenolics and the anti-inflammatory properties of natural products serve in the prevention of neuron inflammation, formation and aggregation of Aβ plaques [5,15,16].Moreover, some natural products also act on the Ach hypothesis by exhibiting inhibitory activity of the acetylcholinesterase enzyme that help ameliorate the cognitive dysfunction induced by AD [6].
Vitex pubescens Vahl (syn.Vitex pinnata Linn), a member of family Lamiaceae [17], is a medium-sized tree with a height ranging from 25 to 30 m [18].It is widely distributed in tropical regions of Asia including Malaysia, Indonesia, the Philippines and Pakistan [19].Vitex pubescens (V.pubescens) is commonly known in Malaysia as "Halban" with ethnopharmacological uses as anti-pyretic, anti-hypertensive, analgesic, wound healing, and for the treatment of gastrointestinal ailments [20,21].Many phytochemical studies were undergone to investigate the phytochemical profile as well as evaluate the biological activities of V. pubescens leaf extracts [20,[22][23][24].Prior studies investigating V. pubescens leaves reported that the abundance of phytochemical classes namely, ecdysteroids, triterpenoids, iridoid glycosides and flavonoid compounds [22,23].Unfortunately, chemical profiling to explore the bioactive metabolites of V. pubescens bark remains insufficient and has not been fully identified despite the long history of its traditional uses.Therefore, this study aimed to investigate the metabolic profile of the total defatted methanol extract of V. pubescens Vahl bark as well as its polar fractions, viz.ethyl acetate and n-butanol fractions for the first time using Ultra-performance liquid chromatography-electrospray ionization-quadrupole time-offlight-tendem mass spectrometry (UPLC-ESI-QTOF-MS/ MS).Moreover, the anti-Alzheimer's disease potential of the total defatted methanolic extract was evaluated invitro using Ellman's microplate assay.

Plant materials
V. pubescens Vahl bark was purchased and authenticated from the herbal company, ETHNO Resources Sdn.Bhd., Malaysia.A voucher specimen (No.PHG-P-VP-302) was deposited in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.

Extraction and fractionation procedures
Four kilograms of V. pubescens Vahl bark powder were firstly defatted with n-hexane (3 × 10 L) using cold maceration method for seven days till exhausting, filtereted and concentrated in-vacuo using a rotatory evaporator (Büchi Labortechnik GmbH, Essen, Germany).The defatted powder was then exhaustively extracted with absolute methanol (7 × 15 L) by cold maceration at ambient temperature for three days, concentrated in-vacuo at 40 °C to yield 185 g of total defatted methanol extract (VT).The VT (180 g) was subjected to successive liquid-liquid fractionation using solvents of increasing polarity, viz.n-hexane (4 × 1 L) followed by DCM (6 × 1 L), ethyl acetate (3 × 1 L) and n-butanol saturated with water (10 × 1 L), concentrated in-vacuo at a temperature below 55 °C till dryness to obtain four different fractions, viz.n-hexane, DCM, ethyl acetate and n-butanol fractions.

Characterization of phytochemicals using UPLC-ESI-QTOF-MS/MS analysis
Metabolites characterization was performed using highresolution Agilent LC-MS system consisting of the Agilent 1290 Infinity II Ultra Performance Liquid Chromatography (UPLC) coupled with Agilent 6545 Electrospray Ionization-quadrupole time-of-flight MS/MS (ESI-QTOF-MS/ MS) using both negative and positive ionization modes.For chromatographic analysis, Kinetex phenyl-hexyl column (1.7 μm, 2.1 × 50 mm) was used.ESI parameters were settled as follows: source voltage at 3.5 kV, capillary temperature at 320 ºC and a sheath gas flow rate of 11 L/ min.Aliquots (1µL) ofthe defatted total methanol extract (VT), ethyl acetate (VE) and n-butanol (VB) fractions were prepared individually as (1 mg/mL MeOH) and each of them was injected on the selected column eluted with a flow rate of 0.4 mL/min.Firstly, the elution was isocratic for one minute with 100% of solvent A (100% H 2 O + 0.1% formic acid) followed by a linear gradient elution for 6 min till 100% solvent B (95% acetonitrile + 5% H 2 O + 0.1% formic acid).The full scan of ions detection was set as following: an isolation width 1.3 ~ m/z, an intensity above 1000 counts at 6 scans/s with 9 selected precursors per cycle and ramped collision energy (5 × m/z/100 + 10 eV) was used.

Total phenolic content determination (TPC)
The total phenolic content for VT was determined spectrophotometrically using the Folin-Ciocalteu method as described by Attard [25,26].The absorbance of the blue complex color was measured at λ max 630 nm spectrophotometrically using a microplate reader FluoStar Omega.The samples were prepared in triplicate, and the mean absorbance value was calculated.The results were represented as mean ± SD.The results were expressed in terms of gallic acid equivalent [27] per milligram of extract (µg GAE/mg extract).The same procedure was repeated for the standard, gallic acid and the calibration curve was constructed as the concentration of 25-100 µg/mL.The mean absorbance of the gallic acid was calculated according to the following equation:

Total flavonoids content determination (TFC)
The total flavonoid content was measured and quantified for VT using aluminum chloride assay which depends on spectrophotometric analysis [28,29].The reaction mixture was prepared and incubated at room temperature for 5 min, after which the yellow color formed was measured at λ max = 420 nm.The same procedure was repeated for the standard, rutin, and the calibration curve Absorbance = 0.0027 gallic acid concentration − 0.0421 R 2 = 0.9994 was constructed at the concentration range of (7.5-1000 µg/mL).The samples were prepared in triplicate, and the mean absorbance value was calculated and represented as mean ± SD.The results were expressed in terms of rutin equivalent (RE) per milligram of extract (µg RE/ mg extract).The mean absorbance of rutin is calculated according to the following equation:

Evaluation of antioxidant activity DPPH free radical scavenging assay
The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay was employed to assess the antioxidant properties of the plant extract.In a 96-well microplate, 100 µL of a freshly prepared 0.1% DPPH solution in methanol was added and mixed with 100 µL of VT in ethanol at different concentrations (15.625, 31.25, 62.5, 125 and 250 µg/mL).The reaction mixture was incubated at room temperature for 30 min then the absorbance of the yellow color intensity was measured at λ max 540 nm spectrophotometrically using a microplate reader FluoStar Omega.Trolox was used as a standard solution prepared with five concentrations ranging from (1.25-12.5 µg/mL).The percentage of DPPH activity inhibition was calculated according to the following equation.
The percent inhibition is plotted against the concentrations and 50% inhibition concentration (IC 50 ) was calculated using Graph pad Prism 6.

Percentage inhibiton =
Average absorbance of blank − average absorbance of the test Average absorbance of blank ×100 methanol to obtain the final ABTS working concentration.In 96 well plate, 190 µL of freshly prepared ABTS reagent was mixed with 10 µL of VT sample (prepared at a concentration of 14 mg/mL in 70% ethanol) or standard stock solution of Trolox (prepared at a concentration of 2 mg/mL).The mixture was then incubated in the dark for 30 min at room temperature.At the end of the incubation period, the reduction of ABTS color intensity was measured at 734 nm using a microplate reader FluoStar Omega.The data was represented as mean ± SD and the percentage inhibition of ABTS radical cation by the extract was calculated using the following equation:

Acetylcholinesterase inhibitory activity using Ellman's microplate assay
The in-vitro assessment of AchE inhibitory activity was evaluated according to Ellman's microplate assay [31].
The samples of VT, VE and VB as well as the standard drug, donepezil were prepared in a set of 8 concentrations (500 -3.9 µg/mL).In a 96 microplate, an aliquot (10 µL) of DTNB (

Characterization of phytochemicals using UPLC-ESI-QTOF/ MS/MS analysis
UPLC-ESI/MS/MS offered a robust and reproducible analytical technique that was employed to tentatively identify and characterize different bioactive secondary metabolites [32].The identification of metabolites was based on comparing the deprotonated/protonated molecular ions and the fragmentation pattern, including the base peak and the major high intensity peaks with those reported in the literature.The UPLC-ESI-QTOF/ MS/MS analysis of the defatted methanol extract of V. pubescens bark and its polar fractions, viz.ethyl acetate and n-butanol fractions revealed the presence of bioactive metabolites belonging to the various mermaid phytochemical classes that were exemplified in polyphenolics such as simple organic acids, phenolic acids, and flavonoids besides the presence of fatty acids and triterpenoids.The order of elution of various chromatographic peaks occurred with decreasing polarity starting with organic acids, and simple phenolics, followed by flavonoid glycosides then aglycones and finally fatty acids and triterpenoids as depicted in Fig. 1.Considering the identified phytochemical metabolites, phenolic acids and flavonoids ionized with higher sensitivity in the negative ion mode, whereas triterpenoids were ionized preferentially in the positive ion mode [33,34].
The detailed identification of the total defatted methanol extract and its ethyl acetate and n-butanol fractions are listed in Table 1.Results revealed a total of 71 metabolites identified in the total defatted methanolic extract.Upon comparing the total defatted methanolic extract with its ethyl acetate and n-butanol fractions, a total of 43 and 55 metabolites have been identified, respectively.Regarding the phytochemical classes of the identified metabolites from total defatted methanol extract and its fractions, it would be conducted that the total defatted methanol extract was enriched with phenolic compounds, viz.organic, phenolic acids (39 metabolites) and flavonoids (21 metabolites) as well as triterpenes (6 metabolites) and fatty acid derivatives (5 metabolites).Whereas for polar fractions, the ethyl acetate fraction   (√) present and (-) absent was enriched with phenolic compounds, viz.organic, phenolic acids (28 metabolites) and flavonoids (15 metabolites) and n-butanol fraction was enriched with phenolic compounds, viz.organic, phenolic acid (29 metabolites) and flavonoids (21 metabolites), also fatty acid derivatives (5 metabolites).The major identified metabolites were illustrated in Fig. 2. A detailed description of the MS/MS spectra of identified metabolities are displayed as Supplementary material in Fig. S1.
From the previous literature, it has been recognized that the only pyrogallol (peak 6) was previously identified from V. negundo leaves and stem [39].Meanwhile, fumaric acid (peak 1), tartaric acid (peak 2), azelaic acid (peak 42), shikimic acid (peak 49) and succinic acid (peak 67) were recognized for the first time in the genus Vitex.

Identification of phenolic acid derivatives
Phenolic acids and their derivatives are one of the most abundant classes of phytochemical metabolites, which account for about a third of all polyphenolic peaks [65].They are organic acids that basically contain a carboxyl group, hydroxyl or methoxy substituent attached directly to an aromatic ring in their chemical structure [66].Phenolic acids are classified into benzoic acid derivatives and Fig. 2 The structures of major identified peaks using UPLC-ESI-QTOF/MS/MS from the defatted methanol extract, ethyl acetate and n-butanol fractions of V. pubescens bark cinnamic acid derivatives [67].In this study, a total of 39 metabolites in the total defatted methanolic extract, 28 metabolites in the ethyl acetate fraction, and 29 metabolites in the n-butanol fraction were tentatively identified as phenolic acids and/or their derivatives.

Benzoic acid derivatives
A total of thirteen benzoic acid derivatives (peaks 5, 8, 9, 10,, 13, 17, 18, 19, 20, 23, 24, and 26) were tentatively identified as either phenolic acids or phenolic acid glycosides from V. pubescens bark.Phenolic acids commonly display a characteristic loss of COO (-44 Da) either in negative or positive ionization modes.Besides, the fragmentation pattern of phenolic acid glycosides could be explained through the loss of intact sugar residue resulting in a base peak fragment ion corresponding to the aglycone part [68].

Identification of flavonoid derivatives
Flavonoids are mainly composed of three-ring diphenyl propane (C 6 C 3 C 6 ) [78].They would present either in aglycone or mostly in glycoside form attached to sugar moiety through a hydroxyl group (flavonoid-O-glycosides) or the anomeric carbon of sugar part attached directly to aglycone part commonly C-6 or C-8 position (flavonoid-C-glycosides) [51].
LC-ESI-MS/MS fragmentation patterns would help in identifying the nature and position of sugar attachment in O-and C-glycosides.The fragmentation of O-glycosides would be easily characterized by the loss of the sugar moiety through cleavage of glycosidic bond yielding the aglycone and sugar parts as product ions.The loss of O-sugar moiety viz.O-hexoside, O-pentoside, O-deoxyhexoside would be revealed through the loss of 162, 132 and 146 Da, respectively [51].

Determination of total phenolic (TPC) and total flavonoid contents (TFC)
The total phenolic and flavonoid contents of the defatted methanol extract of V. pubescens bark were quantified spectrophotometrically as gallic acid and rutin equivalents, respectively, as illustrated in Fig. S2.The total phenolic and flavonoid contents were quantified as 138.61 ± 9.39 µg GAE/mg extract and 119.63 ± 4.62 µg RE/ mg extract, respectively.in comparison with the methanol extract of Vitex agnus castus fruits, the TPC and TFC were found to be 46.50 ± 1.39 µg GAE/mg extract and 10.80 ± 0.26 µg quercetin equivalent /mg extract, respectively [80].Moreover, the ethanol extract of Vitex negundo and Vitex trifolia leaves exhibited TPC of 89.71 mg GAE/g and 77.20 mg GAE/g, respectively, besides, TFC 63.11 mg QE/g and 57.41 mg QE/g [81], .The total defatted methanol extract of V. pubescens exhibited comparatively higher values of TPC and TFC among other Vitex species.These significant values of the total defatted methanol extract would rely on the nature of the identified phytochemicals using UPLC-ESI-MS/MS which revealed the richness of VT with polyphenolic compounds, viz.phenolic acids and flavonoids, especially, di-O-caffeoylquinic acid and flavone-C-glycosides, viz.orientin − 2``-O-rhamnoside (peak 32), orientin (peak 35) and vitexin (peak 38).

DPPH free radical scavenging assay
Antioxidant activity was assessed using DPPH method on the total defatted methanol extract of V. pubescens bark.The VT exhibited promising antioxidant activity with IC 50 value of 52.79 ± 2.16 µg/mL) compared to standard, Trolox (IC 50 7.27 ± 0.309 µg/mL) as illustrated in Fig. 3.In comparison with reported antioxidant activity of genus Vitex, the ethanol extract of the leaves of Vitex negundo and Vitex trifolia exhibited IC 50 values 40.00 and 70.20 µg/mL, respectively [81].The promising antioxidant activity of V. pubescens methanol extract may induce free radical scavenging, neutralization of the lipid-free radicals, and hindering the decomposition of hydroperoxides into free radicals [81,82].Consequently, the detrimental damage induced by oxidative stress would be diminished and potentially delay the progression of Alzheimer's disease [83].Herein, from this approach, V. pubescens extract might be a prominent effector in the treatment of Alzheimer's disease.

Free radical scavenging activity (ABTS) assay
ABTS is one of the commonly applied assays for the determination of radical scavenging activity of the plant extract.It measured the ability of antioxidant compounds to scavenge the ABTS radical cation (ABTS•+), which is generated by the oxidation of ABTS with a strong oxidizing agent [84].The VT exhibited strong free radical scavenging activity (IC 50 value of 10.02 ± 1.039 µg/mL) compared to standard, Trolox (IC 50 5.721 ± 1.023 µg/mL).In comparison with other reported medicinal plants that exhibited antioxidant and anticholinesterase activities, the ethanolic extract of Vitex agnus castus seed exhibited an IC 50 value 12.66 ± 1.25 µg/mL [85].
There are various therapeutic lines for limiting the progression of Alzheimer's disease including the conventional line as AchE inhibitory activity strategy and the recent line as antioxidant treatment [86].Numerous previous studies have linked the use of antioxidant compounds to the reduction of Alzheimer's disease progression, attributing their efficacy to the prevention of oxidative brain damage as well as their anti-amylogenic action [86,87].Based on the aforementioned results, the total defatted methanol extract of V. pubescens recorded high contents of total phenolics and flavonoids (138.61 ± 9.39 µg GAE/mg extract and 119.63 ± 4.62 µg RE/mg extract, respectively, which may be related to its promising antioxidant activity assessed using DPPH (IC 50 52.79± 2.16 µg/mL) and ABTS (IC 50 10.02± 1.039 µg/mL).

Acetylcholinesterase inhibitory activity using Ellman's microplate assay
Ach is a vital neurotransmitter for cognitive function and memory [88,89].The decline in the level of Ach in the brain cells, attributed to the overactivity of the regulatory AchE enzyme, is considered one of the hallmarks of Alzheimer's disease pathogenesis [90,91].Consequently, the elevation of Ach at the synapses of the brain neurons by inhibiting the activity of AchE would be regarded as one of the agreed therapeutic strategies for the treatment of Alzheimer's disease [2].Therefore, screening of the inhibitory activity of the plant extract on AchE level using in-vitro colorimetric Ellman's assay would aid in the discovery of new natural entities for the treatment of Alzheimer's disease [92].The present study assessed the inhibitory activity of the total defatted methanolic extract (VT) of V. pubescens bark as well as its polar fractions, viz. the ethyl acetate (VE) and n-butanol fractions (VB) for the first time as shown in Table 2; Fig. 4. The calculated IC 50 of the total defatted methanolic extract (VT) and its polar fractions, VE and VB were represented in Fig. 5 which exhibited IC 50 values of 52.9, 15.1 and 108.8 µg/Ml, respectively compared to the standard drug, donepezil (IC 50 = 3.89 µg/ mL).The results highlighted the ability of the total defatted Fig. 3 DPPH percent inhibition of V. pubescens extract versus Trolox standard extract of V. pubescens bark and its polar fractions to exert significant inhibitory activity on AchE.Furthermore, VE (IC 50 = 15.1 µg/mL) exhibited the strongest inhibitory activity which were statistically different than standard drug, donepezil (P value < 0.05).Upon comparing to a previous report on other Vitex species, the hydroalcoholic extract of Vitex negundo leaves exhibited IC 50 = 116.00µg/mL displaying lower AchE inhibitory activity than the defatted methanol extract of V. pubescens, whereas it would be in consistence with the inhibitory activity of the n-butanol fraction of V. pubescens bark [93].This promising inhibitory activity could be ascribed to the richness of V. pubescens defatted methanol extract and its polar fractions with diverse polyphenolic compounds with reported Table 2 The inhibitory activity of AchE of the total defatted methanolic extract of V. pubescens bark and its polar fractions using dose response nonlinear regression test.

Conclusion
In this study, a comprehensive characterization of the metabolic profile of the defatted methanol extract of V. pubescens bark as well as its polar fractions, viz.ethyl acetate and n-butanol fractions were performed using UPLC-ESI-QTOF-MS/MS for the first time.A total of 71 metabolites were tentatively identified in the defatted methanol extract.Besides, 43 metabolites were annotated in ethyl acetate fraction and 55 metabolites in the n-butanol fraction.Polyphenolics including phenolic acids, viz.benzoic acid derivatives and acyl quinic acid derivatives, in addition to flavonoids, viz.luteolin-C-glycosides and apigenin-C-glycosides were predominant in V. pubescens defatted methanol extract, ethyl acetate and n-butanol fractions.The metabolic profile shed the light on the potential use of V. pubescens bark for the treatment of Alzheimer's disease as evidenced by the promising in-vitro antioxidant and in-vitro acetylcholinesterase inhibitory activity assessed herein.The current findings provide valuable insights on utilizing V. pubescens total defatted methanol extract and its polar fractions as a natural candidate for the treatment of Alzheimer's disease.

Table 1
Metabolites identified by UPLC-ESI-QTOF/MS/MS analysis of total defatted methanol extract, ethyl acetate, and n-butanol fractions of V.